U.S. patent application number 10/502140 was filed with the patent office on 2005-04-28 for extract of acanthopanax koreanum for the treatment or prevention of hepatitis or the liver protective drug.
Invention is credited to Hong, Young Soo, Kim, Hang Sub, Kim, Young Ho, Lee, Jeong-Hyung, Lee, Jung Joon, Nam, Jeong Bum, Shon, Dong Hwan.
Application Number | 20050089586 10/502140 |
Document ID | / |
Family ID | 27607024 |
Filed Date | 2005-04-28 |
United States Patent
Application |
20050089586 |
Kind Code |
A1 |
Lee, Jung Joon ; et
al. |
April 28, 2005 |
Extract of acanthopanax koreanum for the treatment or prevention of
hepatitis or the liver protective drug
Abstract
The disclosure concerns an extract of Acanthopanax koreanum and
its use. More particularly, it concerns the extract of Acanthopanax
koreanum comprising 1) the extract of Acanthopanax koreanum
extracted from water, 2) among the water extract, the extract of
Acanthopanax koreanum only containing ethanol insoluble part
obtained by precipitating ethanol, 3) among the ethanol insoluble
part, the extract of Acanthopanax koreanum containing polysaccaride
with a molecular weight larger than range of 12,000.about.14,000,
or 4) among the ethanol insoluble part, the extract of Acanthopanax
koreanum containing polysaccaride with a molecular weight larger
than 100,000, which is respectively obtained from the root or stem
of Acanthopanax koreanum. The extract of the present invention
shows a high inhibitory activity against hepatitis and protects the
liver, and thus, can be used for the treatment or prevention of
hepatitis, or as a liver protective drug.
Inventors: |
Lee, Jung Joon; (Oun-dong,
KR) ; Lee, Jeong-Hyung; (Taejeon-si, KR) ;
Hong, Young Soo; (Taejeon-si, KR) ; Kim, Young
Ho; (Taejeon-si, KR) ; Nam, Jeong Bum;
(Taejeon-si, KR) ; Shon, Dong Hwan; (Jeollabuk-do,
KR) ; Kim, Hang Sub; (Taejeon-si, KR) |
Correspondence
Address: |
BACHMAN & LAPOINTE, P.C.
900 CHAPEL STREET
SUITE 1201
NEW HAVEN
CT
06510
US
|
Family ID: |
27607024 |
Appl. No.: |
10/502140 |
Filed: |
July 19, 2004 |
PCT Filed: |
January 24, 2003 |
PCT NO: |
PCT/KR03/00155 |
Current U.S.
Class: |
424/728 ;
514/54 |
Current CPC
Class: |
A61K 36/254 20130101;
A61P 43/00 20180101; A61P 1/16 20180101 |
Class at
Publication: |
424/728 ;
514/054 |
International
Class: |
A61K 035/78; A61K
031/715 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 25, 2002 |
KR |
10-2002-0004553 |
Claims
What is claimed is:
1. Extract of Acanthopanax koreanum containing the water extract
obtained by treating the root or stem of Acanthopanax koreanum with
water, for the treatment or prevention of hepatitis, or as a liver
protective drug.
2. The extract of claim 1, wherein the extract contains the ethanol
insoluble part obtained by treating the said water extract with
ethanol, among the said water extract obtained from the root or
stem of Acanthopanax koreanum.
3. The extract of claim 2, wherein extract contains the fraction
containing polysaccharide with a molecular weight larger than range
of 12,000.about.14,000, among the said ethanol insoluble part
obtained from the root or stem of Acanthopanax koreanum.
4. The extract of claim 2, wherein extract contains the fraction
containing polysaccharide with a molecular weight larger than
100,000, among the said ethanol insoluble part obtained from the
root or stem of Acanthopanax koreanum.
5. The extract of claim 2, wherein extract contains the said
ethanol insoluble part of which the final concentration of ethanol
is in between 50 and 90%.
6. The extract of claim 2, wherein extract contains the said
ethanol insoluble part of which the final concentration of ethanol
is 80%.
7. Therapeutic agent or preventor of hepatitis containing the
extract of Acanthopanax koreanum of any of claim 1 to claim 4 as an
effective ingredient.
8. Liver protective drug containing the extract of Acanthopanax
koreanum of any of claim 1 to claim 4 as an effective
ingredient.
9. TNF-.alpha. inhibitor containing the extract of Acanthopanax
koreanum of any of claim 1 to claim 4 as an effective
ingredient.
10. Health supplementary food containing the extract of
Acanthopanax koreanum of any of claim 1 to claim 4 as an effective
ingredient.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to an extract of Acanthopanax
koreanum for the treatment or prevention of hepatitis or the liver
protective drug and its use.
BACKGROUND OF THE INVENTION
[0002] Located between the digestive system and body circulating
system in human body, liver plays an important role in defending
our body from the harmful intrusion of toxic substances and in
metabolism. Since foreign substances intruded into human body first
passes through the liver, liver has a high possibility to be
exposed to various toxic substances other than nutrients, and thus,
has a higher possibility to be injured than other internal
organs.
[0003] As an internal organ with excellent restoration ability,
liver can completely recover its functions in slight damages.
However, if liver is continuously damaged by alcohol over
ingestion, chemical substance abuse, viral hepatitis, and bile
secretion suspension, not only its functions are deteriorated, but
a part of liver tissues are completely damaged, and the thus
damaged part cannot be completely restored, which goes through
liver fibrosis and may finally advance into fatal cirrhosis.
Further, liver diseases do not show any pains or subjective
symptoms at the initial stage, but they are found at the terminal
stage. Therefore, it is impossible to treat liver diseases at a
proper stage, and thus, liver diseases show a high death rate.
[0004] Regardless of the severity of liver diseases, an effective
liver-disease therapeutic has not yet been found. As for liver
diseases caused by viral hepatitis, anti-virus drugs are being
used, but their side effects cause serious problems. As for liver
diseases caused by toxic substances recently increasing due to
alcohol and environment pollution, an effective liver disease
therapeutic has not yet been found. Accordingly, the development of
a drug, which treats and prevents liver damage while maintaining
the structure and function of liver tissue is keenly required.
However, since no experimental method has been developed till now,
there are many limitations in developing a liver disease
therapeutic. That is, in fact, there is not enough experimental
support on the drugs referred to as liver protective drugs.
[0005] However, recently, an animal model has been developed which
contributed to the development of a liver disease therapeutic. In
this connection, an animal model induced with carbon tetrachloride
is used in order to develop a liver disease therapeutic caused by
toxic substances, and an acute hepatitis model induced with
D-galactosamine (hereinafter abbreviated into "D-GalN") and
lipopolysaccharide (hereinafter abbreviated into "LPS") are used in
order to develop a liver disease therapeutic caused by virus.
[0006] Especially, since the above liver damage model induced with
D-GalN/LPS causes liver damage by the immune reaction which is
actually proceeded in most liver diseases, it is the animal model
appropriate for the treatment and prevention of liver diseases
[Ken-Ichiro Kosai, Kunio Matsumoto, Hiroshi Funakoshi and Toshikazu
Nakamura, Hepatocyte Growth factor Prevents Endotoxin-induced
Lethal Hepatic Failure in Mice. Hepatology, 1999, 30, 151-159]. In
acute hepatitis model induced with D-GalN/LPS, D-GalN inhibits RNA
synthesis and protein synthesis in cells to maximize liver toxicity
caused by LPS, and LPS promotes the secretion and synthesis of
cytokine, nitrogen monoxide (NO) and active oxygen of the kupffer
cell, which is the macrophage of liver. It has been found out that
tumor necrosis factor alpha (TNF-.alpha.) induced by excessive
nitrogen monoxide is a main etiological agent of septicemia or
acute hepatitis. In fact, it has been found out that TNF-.alpha.
causes in vivo and in vitro hepatocyte death [Michael D Josephs, F.
Rena Bahjat, Kunitaro Fukuzuka, Riadh Ksontini, Carmen C.
Solorzano, Carl K. Edwards III, Cynthia L. Tannahill, Sally L. D.
MacKAY, Edward M. Copeland III, and Lyle L. Moldawer.
Lipopolysaccharde and D-galactosamine-induced hepatic injury is
mediated by TNF-.alpha. and not by Fas ligand. Am J Physiol
Regulatory Integrative comp Physiol, 2000, 278, R1196-R1201].
Further, Leist considers TNF-.alpha. to be the most important
factor in causing liver damage by proving that the mortality is
decreased when the acute liver damage model induced with D-GalN/LPS
is treated with anti-tumor necrosis factor alpha (anti-TNF-.alpha.)
[Leist M. Gauntner F., Bohlinger I, Germann P G, Tiegs G, Wendal A.
Murine hepatocytee apoptosis induced in vitro and in vivo by
TNF-.alpha. requires transcriptional arrest. J. Immunol. 1994, 153,
1778-1788].
[0007] The cell death process is largely affected by Bcl-2 family
(pro- and anti-apoptotic member) proteins, which can be exemplified
by Bax protein or Bid protein [Yongge Zhao, Shuchen Li, Erin E.
Childs, Diane K. Kuharsky, and Xiao-Ming Yin. Activation of
Pro-death Bcl-2 Family Proteins and Mitochondria Apoptosis Pathway
in Tumor Necrosis Factor-a-induced Liver Injury. J. Biol. Chem.
2001, 276, 27432-27440].
[0008] More particularly, the death process of hepatocyte activates
caspase 8 by interacting with FADD or TRADD protein having a death
domain, when proteins inducing apoptosis such as TNF bind to the
cell receptor, TNF receptor 1. The thus activated caspase 8 cleaves
Bid protein and transforms it into an activated form, tBid. The
thus transformed tBid is translocated to mitochondria to cause
cytchrome C release. The thus released cytchrome C activates
pro-caspase 9 into caspase 9, and this caspase 9 induces the
cooperative effects of lower caspases by activating caspase 3 which
leads all cells to apoptosis [Xiao-Ming Yin, Bid, a critical
mediator for apoptosis induced by the activation of Fas/TNF-R1
death receptors in hepatocytes. J. Mol, 2000, 78, 203-211].
[0009] Therefore, hepatocyte apoptosis generated in acute
liver-injury model induced with D-GalN/LPS causes the activation of
apoptosis pathway by TNF-.alpha. receptor. Accordingly, it can be
proved that an extract of the stem or root of Acanthopanax koreanum
activates liver protection by the said working by examining whether
Acanthopanax koreanum polysaccharide inhibits TNF-.alpha. activity
itself, and by proving that Acanthopanax koreanum polysaccharide
inhibits the expression of the important protein activated by
TNF-.alpha..
[0010] In addition, the amount of circulating alanine
aminotransferase (hereinafter abbreviated into ALT, GPT index) and
aspartate aminotranasferase (hereinafter abbreviated into AST, GOT
index), and the concentration of circulating tumor necrosis factor
(TNF-.alpha.) are measured to determine liver protection activity
in the acute liver damage model induced with D-GalN/LPS. In
addition, the liver protection effect of the sample can be
determined more precisely by measuring apoptosis inhibition effect
of hepatocyte using the activity inhibiting hepatocyte DNA cleavage
as an index, and by measuring the 24 hour survival rate of the
mouse.
[0011] Recently a drug for the treatment or prevention of hepatitis
by protecting the liver functions by using the said animal model is
being developed. Especially, it has been reported that saponin,
bupleuroside compounds (H. Maysuda et al., Bioorg. Med. Chem.,
1997, 7, 2193-2198), naringin (K. Kawaguchi et al., Eur. J.
Pharmacol., 1999, 368, 245-250), green tea extract(P. HE et al., J.
Nutr., 2001, 131, 1560-1567), polysaccharides extracted from the
seeds of Celosia argentea show activity in protecting the liver
functions in the liver damage model induced with D-GalN/LPS and
inhibiting the experimental animal lethality (K. Hase et al., Biol,
Pharm. Bull., 1996, 19, 567-572).
[0012] In addition, there is a report on the liver protection
activity of an extract of Acanthopanax senticosus [Chun-Ching Lin
and Pei-Chen Huang, Phytotherapy Research, 2000, 14, 489-494].
However, no specific examples on an extract of Acanthopanax
koreanum for the treatment or prevention of hepatitis or liver
protection have been reported. Acanthopanax senticosus
morphologically differs a lot from Acanthopanax koreanum.
Acanthopanax senticosus is thickly wooded with thin long thorns on
its bark and branches, and the style of the fruit is divided into
5. Further, it is mainly distributed in the alpine regions of
Korea; Hokkaido, Japan; the Heilung Riverside, China; and Siberia,
Russia. Acanthopanax koreanum is wooded with triangle shaped
grayed-brown thorns with a large base, and the style of its fruit
is divided into 2. Further, it is a Korean indigenous plant
distributed in the southern part of Korea including Chejudo.
[0013] Further, Acanthopanax koreanum comprises acanthoic acid;
pimara-9(11)-dien-19 oic acid as its main component, whereas,
Acanthopanax senticosus does not comprise such component [Young H.
Kim and Bo S. Chung J. Nat. Prod. 1988, 51 1080-1083].
[0014] Therefore, in connection with liver diseases caused by
hepatitis virus and toxic substances, inventors have devoted
themselves in developing a liver disease therapeutic with less side
effects, and based on the working model of the acute liver-injury
induced with D-GalN/LPS the present invention has been completed by
proving an accurate experimental method and results that an extract
from the root or stem of Acanthopanax koreanum is effective on the
treatment and prevention of liver damage while maintaining the
structure and function of the liver tissues.
SUMMARY OF THE INVENTION
[0015] It is an object of the present invention to provide an
extract of Acanthopanax koreanum for the treatment or prevention of
hepatitis or the liver protective drug.
[0016] It is another object of the present invention to provide a
use wherein an extract of Acanthopanax koreanum can be used for the
treatment or prevention of hepatitis or as a liver protective drug
on the basis of the working model of the acute liver-injury mice
induced with D-GalN/LPS.
BEST MODE FOR CARRYING OUT THE INVENTION
[0017] FIG. 1 shows HPLC analysis of the fraction containing
polysaccharide with a molecular weight larger than range of
12,000.about.14,000, among the ethanol insoluble part, obtained
from the root of Acanthopanax koreanum
[0018] FIG. 2 shows HPLC analysis of the fraction containing
polysaccharide with a molecular weight larger than range of
12,000.about.14,000, among the ethanol insoluble part, obtained
from the stem of Acanthopanax koreanum.
[0019] FIG. 3 shows effect of the extract obtained from the root of
Acanthopanax koreanum, on the survival of mice in liver-injury
model induced by D-GalN/LPS.
[0020] a group treated with the water extract of Acanthopanax
koreanum root (hereinafter abbreviated into "SRW", 300 mg/kg);
[0021] a group treated with the 80%-ethanol insoluble part of the
water extract of Acanthopanax koreanum root (hereinafter
abbreviated into "SRWB", 300 mg/kg); and
[0022] .circle-solid. a group treated with physiological saline
solution
[0023] FIG. 4 shows effect of the extract obtained from the stem of
Acanthopanax koreanum, on the survival of mice in liver-injury
model induced by D-GalN/LPS
[0024] a group treated with the water extract of Acanthopanax
koreanum stem (hereinafter abbreviated into "SSW", 300 mg/kg);
[0025] a group treated with the 80%-ethanol insoluble part of the
water extract of Acanthopanax koreanum stem (hereinafter
abbreviated into "SSWB", 300 mg/kg); and
[0026] .circle-solid. a group treated with physiological saline
solution
[0027] FIG. 5 shows effect of the fraction containing
polysaccharide with a molecular weight larger than 100,000, among
the said 80%-ethanol insoluble part obtained from the stem of
Acanthopanax koreanum, on the survival of mice in liver-injury
model induced by D-GalN/LPS.
[0028] .box-solid. a group treated with the fraction containing
polysaccharide with a molecular weight larger than 100,000,
obtained from the stem of Acanthopanax koreanum (30 mg/kg);
[0029] .tangle-solidup. a group treated with the fraction
containing polysaccharide with a molecular weight larger than
100,000, obtained from the stem of Acanthopanax koreanum group (100
mg/kg); and
[0030] .diamond-solid. a group treated with physiological saline
solution
[0031] FIG. 6 shows effect of the fraction containing
polysaccharide with a molecular weight larger than 100,000, among
the said 80%-ethanol insoluble part obtained from the root of
Acanthopanax koreanum on the survival of mice in liver-injury model
induced by D-GalN/LPS.
[0032] .quadrature. a group treated with the fraction containing
polysaccharide with a molecular weight larger than 100,000,
obtained from the root of Acanthopanax koreanum (30 mg/kg)
[0033] .tangle-solidup. a group treated with the fraction
containing polysaccharide with a molecular weight larger than
100,000, obtained from the root of Acanthopanax koreanum (100
mg/kg);and
[0034] .diamond-solid. a group treated with physiological saline
solution
[0035] FIG. 7 shows effect of the extract obtained from the root or
stem of Acanthopanax koreanum, on the survival of mice in
liver-injury model induced by D-GalN/TND-.alpha..
[0036] .quadrature. a group treated with the fraction containing
polysaccharide with a molecular weight larger than 100,000,
obtained from the stem of Acanthopanax koreanum (100 mg/kg)
[0037] .tangle-solidup. a group treated with the fraction
containing polysaccharide with a molecular weight larger than
100,000, obtained from the root of Acanthopanax koreanum (100
mg/kg);and
[0038] .diamond-solid. a group treated with physiological saline
solution
[0039] FIG. 8 shows effect of the extract on the DNA fragmentation
of liver cell in liver-injury model induced by D-GalN/LPS.
[0040] Marker: comparative marker
[0041] 1: DNA isolated from the liver of the mice to which
physiological saline solution was administered.
[0042] 2: DNA isolated from the liver of the mice to which
physiological saline solution was administered after administration
of D-GalN/LPS.
[0043] 3: DNA isolated from the liver of the mice to which
D-GalN/LPS and 300 mg/kg of the water extract obtained from the
root of Acanthopanax koreanum were administered.
[0044] 4: DNA isolated from the liver of the mice to which
D-GalN/LPS and 300 mg/kg of the water extract obtained from the
stem of Acanthopanax koreanum were administered.
[0045] 5: DNA isolated from the liver of the mice to which
D-GalN/LPS and 300 mg/kg of the 80%-ethanol insoluble part of the
water extract obtained from the stem of Acanthopanax koreanum were
adminstered.
[0046] 6: DNA isolated from the liver of the mice to which
D-GalN/LPS and the fraction containing polysaccharide with a
molecular weight larger than range of 12,000.about.14,000, obtained
by dialyzing the said 80%-ethanol insoluble part were
administered.
[0047] FIG. 9 shows effect of the fraction containing
polysaccharide with a molecular weight larger than 100,000, among
the ethanol insoluble part on the expression of pro-apoptotic
protein;
[0048] A: the extract obtained from the stem of Acanthopanax
koreanum
[0049] B: the extract obtained from the root of Acanthopanax
koreanum
[0050] FIG. 10 shows effect of the oral administration of the
80%-ethanol insoluble part of the water extract obtained from the
stem of Acanthopanax koreanum on the liver protection.
[0051] FIG. 11 shows effect of the oral administration of the
ethanol insoluble part of the water extract obtained from the root
of Acanthopanax koreanum on the liver protection.
[0052] .tangle-solidup.: a group treated with the 80%-ethanol
insoluble part of the water extract obtained from the stem or root
of Acanthopanax koreanum (30 mg/kg)
[0053] : a group treated with the 80%-ethanol insoluble part of the
water extract obtained from the stem or root of Acanthopanax
koreanum (100 mg/kg);and
[0054] .diamond-solid. a group treated with physiological saline
solution
[0055] The present invention provides an extract of Acanthopanax
koreanum for the treatment or prevention of hepatitis, or as a
liver protective drug.
[0056] The extract of the present invention provides 1) the water
extract of the root or stem of Acanthopanax koreanum 2) the ethanol
insoluble part among the said water extract, obtained by treating
the said water extract with ethanol, 3) the fraction containing
polysaccharide with a molecular weight larger than range of
12,000.about.14,000, among the said ethanol insoluble part, or 4)
the fraction containing polysaccharide with a molecular weight
larger than 100,000, among the said ethanol insoluble part.
[0057] Furthermore, the present invention provides a use of the
extract obtained from the root or stem of Acanthopanax koreanum, as
a therapeutic agent for treatment or preventer of hepatitis, or a
liver protective drug.
[0058] The extract of the present invention inhibits activity of
TNF-.alpha. and expression of important protein activated by
TNF-.alpha. in acute liver-injury mice model induced by D-GalN/LPS.
Also, the extract of the present invention made high survival rate
maintained in the experiment for measurement of lethality rate
progressed for 24 hours. Therefore, based on the working model of
the acute liver-injury mice induced by D-GalN/LPS, the extract of
the present invention can be used as a therapeutic agent or
preventer of the hepatitis, or a liver protective drug.
[0059] The present invention uses Acanthopanax koreanum of which
the place of origin is KOREA. The common extract of Acanthopanax
koreanum is prepared by treating the root or leaf of Acanthopanax
koreanum with solvent. Furthermore, to apply Acanthopanax koreanum
to experimental or drug material, it must be withered up. However,
in the present invention, the extract of Acanthopanax koreanum
obtained from the root of the Acanthopanax koreanum has the same
efficiency as one obtained from the stem thereof. Therefore,
Acanthopanax koreanum does not have to be withered up.
[0060] The said water extract of Acanthopanax koreanum is prepared
by treating the root or stem of Acanthopanax koreanum with water by
the method such as dipping, maceration or heating. Preferably, the
said water extract is prepared by heating the root or stem of
Acanthopanax koreanum at the temperature higher than 90.degree.
C.
[0061] The ethanol insoluble part of the said water extract is
prepared by treating the said water extract of Acanthopanax
koreanum with ethanol, of which the final concentration of ethanol
is 50.about.90%. Preferably, the concentration is 75.about.85%,
more preferably 80%.
[0062] The fraction containing polysaccharide with a molecular
weight larger than range of 12,000.about.14,000, or larger than
100,000, among the said ethanol insoluble part is prepared by
dialysis of the said extract through dialyzing diaphagram or filter
membrane corresponding to the molecular weight, respectively.
[0063] As shown in the experiment using liver injury mice model
induced by D-GalN/LPS, the said water extract of Acanthopanax
koreanum of the present invention, preferably the said ethanol
insoluble part, more preferably the fraction containing
polysaccharide with a molecular weight larger than range of
12,000.about.14,000, or larger than 100,000, among the said ethanol
insoluble part has an excellent efficiency for the treatment or
prevention of hepatitis, or a liver protective drug.
[0064] FIG. 3 or 4 shows the lethality rate of the mice induced by
D-GalN/LPS, measured for 24 hours when the extract of Acanthopanax
koreanum is administered to the said mice. More particularly, the
said water extract and the said 80%-ethanol insoluble part shows
excellent survival rate by more than 70.about.80% in 24 hours
without liver toxicity. Therefore, the extract has a high efficacy
such as liver protection in the mice model of the acute hepatitis.
Also, FIG. 5 and FIG. 6 show that the fraction containing
polysaccharide with a molecular weight larger than 100,000, among
the ethanol insoluble part obtained from the root or stem of
Acanthopanax koreanum maintains the survival rate higher than 90%,
respectively. Especially, the fraction obtained from the root of
Acanthopanax koreanum has an efficiency that when 30 mg/kg or 100
mg/kg of the said fraction was administered to the mice, the
survival rate is maintained to the extent of 100%. Therefore, the
fraction has an excellent efficacy for the liver protection.
[0065] FIG. 8 shows that the said ethanol insoluble part prepared
by treating the said water extract with ethanol, of which the final
concentration of ethanol is 80 and the fraction containing
polysaccharide with a molecular weight larger than range of
12,000.about.14,000, among the ethanol insoluble part inhibit DNA
fragmentation in liver cell.
[0066] FIG. 9 shows that the fraction containing polysaccharide
with a molecular weight larger than 100,000, among the said ethanol
insoluble part influences the expression of pro-apoptotic protein.
Particularly, the result shows that the said fraction used in the
experiment inhibits the expression of Bax protein concentration
dependently. Also, in case that pro-apoptotic protein is Bid
protein, experimental group to which the fraction containing
polysaccharide with a molecular weight larger than 100,000 is
administered, has a similar inhibition relative to the control
group concentration dependently. Therefore, the said fraction
containing polysaccharide with a molecular weight larger than
100,000 inhibits the expression of pro-apoptotic protein activated
by TNF-.alpha. in liver cell.
[0067] Also, FIG. 10 and FIG. 11 show that when the 80%-ethanol
insoluble part prepared by treating the water extract with ethanol
is orally administered to the mice, the survival rate of the mice
is maintained to the extent of 50% for 24 hours. However, the
control group to which the said part is not administered is
deceased within 12 hours.
[0068] Therefore, based on the working model of the acute
liver-injury mice induced by D-GalN/LPS, the extract of the present
invention maintains the serum level of TNF-.alpha., the cause of
acute hepatitis, to be similar to level of the normal group. Also,
the said extract inhibits the expression of pro-apoptotic protein
activated by TNF-.alpha.. Also, the said extract reduces the serum
level of AST or ALT to the similar to that of normal group.
Therefore, the said extract has an excellent efficacy for treatment
or prevention of hepatitis, or a liver protection.
[0069] The extract of Acanthopanax koreanum of the present
invention can be used for health supplementary food.
[0070] The extract of the present invention can be administered in
various methods in an amount of dosage. The said composition
contains pharmaceutically acceptable carrier. More particularly,
any of pharmaceutically acceptable carrier selected from sterilized
solution, or the standard carrier used in the known formulation,
such as tablet, coating tablet and capsule can be used.
Conservatively, carrier is selected from diluting agent containing
starch, milk, glucose, clay, gelatin, stearic acid, talc, vegetable
oil, gum and glycol, or the known diluting agents. Also, the said
carrier can be selected from flavoring agent, pigment or another
components.
[0071] To administer pharmaceutical composition containing the
extract of the present invention as an effective ingredient within
the said dosage, administrations can be accomplished by the
conservative methods such as oral administration, intravenous
injection, intramuscular injection or transdermal administration,
but is not limited to them. In clinical application, the
formulation can be administered through the oral or parenteral
administration. The said formulation is prepared by using common
diluent containing packing agent, bulking agent, bindng agent,
moistening agent, disintegrating agent, surfactant or diluting
agent.
[0072] Composition for oral administration can be formulated into
the solid formulation such as tablet, pill, powder, granule or
capsule. The said formulation is prepared by mixing one or more
selected from the extract of the present invention with diluting
agent for example, starch, calcium carbonate, sucrose, lactose or
gelatin. Also, in addition to simple diluting agent, lubricant such
as magnesium stearate is used.
[0073] Composition for oral administration can be formulated into
the liquid formulation such as suspension, liquid, emulsion, syrup.
The said formulation contains various diluting agent for example,
moistening agent, flavoring agent, aromatic agent or preservative,
in addition to simple diluent, for example water, liquid paraffin.
Preferably, the composition can be formulated into the tablet,
capsule, or drink, and used as medicine or health supplementary
food.
[0074] Also, the pharmaceutical composition of the present
invention can be parenterally administered. The formulation for
parenteral administration is prepared by mixing one or more
selected from the extract of the present invention with stabilizer
or buffer in water, preparing into solution or suspension, and
formulating into an unit dosage form such as ample or vial.
[0075] The extract used for therapeutic agent or preventor of
hepatitis, or a liver protective drug is preferably the ethanol
insoluble part, more preferably the fraction containing
polysaccharide with a molecular weight larger than range of
12,000.about.14,000 among the said ethanol insoluble part. However,
in the process of purification using dialyzing diaphagram, yield is
30.about.40%. Therefore, preferably, the ethanol insoluble part is
used. An effective ingredient of the present invention can be
administered through one or many times per 1 day. The dosage of the
effective ingredient is preferable 1.about.1000 mg/kg/day, more
preferable 10.about.1000 mg/kg/day, depending upon absorptivity of
the active component in vivo, activity, excretion rate, age, sex
and state of the patients, seriousness of disease under treatment.
More particularly, the preferable dosage of the water extract, the
70%-ethanol insoluble part, and the fraction containing
polysaccharide with a molecular weight larger than range of
12000.about.14000, among the said ethanol insoluble part is
300.about.1000 mg/kg/day, 100.about.500 mg/kg/day and 10.about.300
mg/kg/day, respectively. Accurate dosage, method and frequency for
administration were selected according to property of the
formulation, weight and state of administrative group, or
characteristic of derivatives used.
[0076] Toxic test for the mice shows that the extract of the
present invention, obtained from the root or stem of Acanthopanax
koreanum is nontoxic. Also, the test shows that LD50 is higher than
2000 mg/kg, the extract of the present invention has high safety
and stability. Therefore, the extract of the present invention, as
a liver protective drug can be safely administered to the body.
[0077] The present invention will be explained in more detail with
reference to the following examples. However the following examples
are provided only to illustrate the present invention, and the
present invention is not limited to them.
EXAMPLE 1
[0078] Preparation of the Water Extract of Acanthopanax
koreanum
[0079] The root or stem of Acanthopanax koreanum is dried and
sliced to small pieces, respectively. In a 10 L flask, 1 kg of
slices was mixed with water and extracted at the temperature more
than 90.degree. C. for 3 hours. The extraction is repeated two
times. The extract is filtered through the filtrate memberane,
concentrated under reduced pressure and lyophilized to prepare the
water extract. Quantity of the water extract obtained from the root
or stem of Acanthopanax koreanum is 142 g or 80 g,
respectively.
EXAMPLE 2
[0080] Preparation of the Ethanol Insoluble Part of Acanthopanax
koreanum
[0081] The ethanol insoluble part containing more polysaccharide
relative to the said water extract was prepared by treating the
said water extract with ethanol. 20 g of the said water extract
obtained from the root or stem of Acanthopanax koreanum was
dissolved in 50 ml of water. Ethanol was added to the solution, of
which final concentration of ethanol was 60.about.80%. The solution
was centrifuged to acquire the precipitated insoluble part. The
insoluble part was dried.
[0082] Also, the ethanol filtrate after centrifugal separation was
concentrated to acquire the ethanol extract from the said water
extract obtained from the root or stem of Acanthopanax koreanum,
respectively. Quantity of the ethanol insoluble part and ethanol
extract obtained by treating the water said extract of the root or
stem of Acanthopanax koreanum with ethanol was shown in the table
1.
1TABLE 1 Quantity of the ethanol insoluble part and the ethanol
extract Final Quantity of Conc. of the water Root Stem ethanol
extract(g) SRWB.sup.a(g) SRWS.sup.b(g) SSWB.sup.c(g) SSWS.sup.d(g)
60% 20 4.5 15.3 3.7 16.5 70& 20 6.9 13.5 5.1 15.2 80% 20 8.6
11.4 5.6 14.9 .sup.athe ethanol insoluble part of the water extract
obtained from the root of Acanthopanax koreanum(hereinafter,
abbreviated into SRWB) .sup.bthe ethanol soluble part of the water
extract obtained from the root of Acanthopanax
koreanum(hereinafter, abbreviated into SRWS) .sup.cthe ethanol
insoluble part of the water extract obtained from the stem of
Acanthopanax koreanum(hereinafter, abbreviated into SSWB) .sup.dthe
ethanol soluble part of the water extract obtained from the stem of
Acanthopanax koreanum(hereinafter, abbreviated into SSWS)
[0083] As shown in the table 1, in case of treating the water
extract with ethanol, the yield of ethanol insoluble part was
higher when final concentration of ethanol is 80% than when final
concentration of ethanol is 60% or 70%.
EXAMPLE 3
[0084] Preparation of the Fraction Containing Polysaccharide with a
Molecular Weight Larger than Range of 12,000.about.14,000, Among
the said Ethanol Insoluble Part
[0085] The ethanol insoluble part obtained by treating the water
extract with ethanol has polysaccharide as the major component.
However, to purify the said polysaccharide, the fraction containing
polysaccharide with a molecular weight larger than range of
12000.about.14000 is prepared by dialysis of the said extract
through dialyzing diaphagram (Spectra Por Spectrum Medical
Industries Inc. Houston Tex.) corresponding to the molecular
weight, respectively.
[0086] The water extract obtained from the root or stem of
Acanthopanax koreanum was treated with 80% ethanol to prepare the
ethanol insoluble part, respectively. The 500 mg of the said
ethanol insoluble part was dissolved in 7 ml of distilled water,
thereafter the solution was centrifuged to acquire the supernatant.
The said supernatant was dialysed through the dialyzing diaphagram
passing the compound with a molecular weight in the range of
12000.about.14000, thereafter filter cake was lyophilized. Quantity
of the fraction containing polysaccharide with a molecular weight
larger than range of 12000.about.14000, obtained from the 300 mg of
the ethanol insoluble part prepared from the root or stem of
Acanthopanax koreanum is 130 mg or 120 mg, respectively.
EXAMPLE 4
[0087] Preparation of the Fraction Containing Polysaccharide with a
Molecular Weight Larger than 100,000, Among the said Ethanol
Insoluble Part
[0088] To purify the polysaccharide of Acanthopanax koreanum
additionally, the fraction containing polysaccharide with a
molecular weight larger than 100,000 is prepared by filtering the
said extract through dialyzing diaphagram (Spectra Por Spectrum
Medical Industries Inc. Houston Tex.) passing the compound with a
molecular weight smaller than 100,000.
[0089] The water extract obtained from the root or stem of
Acanthopanax koreanum was treated with ethanol to prepare the
ethanol extract. 15.5 g or 20.7 g of the ethanol extract was
dissolved in the water, respectively. The solution was dialysed
through the dialyzing diaphagram passing the compound with a
molecular weight smaller than 100,000, thereafter filter cake was
lyophilized. Yield of the fraction containing polysaccharide with a
molecular weight larger than 100,000, obtained from the ethanol
extract prepared from the root or stem of Acanthopanax koreanum is
12% or 10.2%, respectively.
[0090] The experiment confirming that the extract of the present
invention has efficiency for treatment of hepatitis or liver
protection in liver-injury mice model induced by D-GalN/LPS was
accomplished as shown in the below.
EXPERIMENTAL EXAMPLE 1
[0091] HPLC Analysis of the Fraction Containing Polysaccharide
[0092] As for the fraction containing polysaccharide with a
molecular weight larger than range of 12,000.about.14,000, obtained
from the said ethanol insoluble part, molecular weight or number of
the polysaccharide was measured.
[0093] The fraction containing polysaccharide with a molecular
weight larger than range of 12,000.about.14,000, obtained from the
root or stem of Acanthopanax koreanum was dissolved in the
distilled water, of which the final concentration of polysaccharide
was 10 mg/ml. 20 .mu.l of the solution was injected to YMC-pack
Diol-300 column (YMC Co. Kyoto, Japan). The elution rate of eluent
is 1 ml/min. Molecular weight and number of the polysaccharide in
the sample were analyzed by evaporation light scattering detector
(Alltech 500 ELSD) (shown in FIG. 1 or FIG. 2).
[0094] The extract containing polysaccharide, obtained from the
stem of Acanthopanax koreanum is mainly comprised of polysaccharide
with a molecular weight 900,000. Also, polysaccharide with a
various molecular weight, for example, 450,000 or 250,000 was
contained in the extract. Also, polysaccharide with a molecular
weight in the range of 14,000.about.200,000 was contained in the
extract. The extract containing polysaccharide, obtained from the
root of Acanthopanax koreanum is mainly comprised of polysaccharide
with a molecular weight more than 1,000,000. Also, polysaccharide
with a various molecular weight, for example, 2,000,000, 450,000 or
300,000 was contained in the extract. More polysaccharide with a
molecular weight in the range of 14,000.about.200,000 was contained
in the extract of the stem than that of the root.
EXPERIMENTAL EXAMPLE 2
[0095] The Serum Level of AST or ALT in Liver-Injury Mice Model
Induced by D-GalN/LPS
[0096] The experiment confirming that the extract of the present
invention has a efficacy for prevention of hepatitis in
liver-injury mice model induced by D-GalN/LPS by measuring the
serum level of AST or ALT was accomplished as shown in the
below.
[0097] After mice (B57BL/6) having body weights of 20 g were
adjusted to a new environment for 1 week, they were used in the
experiment. Mice were fully fed before the experiment. The
experiment was accomplished with mice divided to three groups such
as normal group, group to which physiological saline solution was
administered (physiological saline solution-treated group), group
to which the extract was administered(the extract-treated group).
The said extract was selected from the group consisting of 1) the
methanol extract obtained by treating the root or stem of
Acanthopanax koreanum with 100% methanol, 2) the water extract of
Acanthopanax koreanum obtained by treating the root or stem of
Acanthopanax koreanum with water, 3) the ethanol extract obtained
by treating the root or stem of Acanthopanax koreanum with 70%
ethanol 4) the ethanol insoluble part among the said water extract,
obtained by treating the said water extract with ethanol, of which
final concentration of ethanol is 80%, or 5) the fraction
containing polysaccharide with a molecular weight larger than range
of 12,000.about.14,000 among the ethanol insoluble part.
[0098] The said extract dissolved in physiological saline solution
was intraperitoneally administered to the said mice model twice in
an amount of 50 mg/kg or 300 mg/kg, respectively, at 12 and 1 hour
earlier than D-Gal and LPS treatment. Thereafter, D-Gal and LPS
were subsequently administered to the said mice model in an amount
of 700 mg/kg and 10 mg/kg, respectively.
[0099] Physiological saline solution was administered to the
control group of the said mice model in the same amount. Blood was
collected from the mice at 8 hours after administration. DNA was
isolated from the part of liver tissue. The part of liver was
maintained in 10% formalin for staining of tissue. The collected
blood was centrifuged at 3000 rpm, the serum was isolated and
stored at -20.degree. C. The serum level of AST and ALT was
measured by using kit purchased from the ARKARY FACTORY (JAPAN) and
auto-dry chemistry analyzer (SPOTCHEM, ARKARY, JAPAN) to measure
the serum level of GOT and GPT in blood. The result was shown in
table 2 and 3.
2TABLE 2 The effect of the various root extract of Acanthopanax
koreanum on the serum level of AST and ALT in liver-injury mice
model induced by D-GalN/LPS Amount Group (mg/kg) AST ALT Normal
group -- 67 .+-. 11 23 .+-. 11 Physiological saline -- 3456 .+-.
1064 3678 .+-. 1291 solution-treated group (control group) The
water extract-treated 50 567 .+-. 181 548 .+-. 139 group 300 190
.+-. 40 117 .+-. 47 The 70% ethanol extract- 50 678 .+-. 125 598
.+-. 217 treated group 300 228 .+-. 45 96 .+-. 33 Treatment The
ethanol 50 378 .+-. 113 364 .+-. 128 of the insoluble part- 300 189
.+-. 45 143 .+-. 53 water treated group extract The ethanol 50 589
.+-. 139 543 .+-. 145 with soluble part- 300 450 .+-. 120 425 .+-.
107 ethanol treated group The fraction containing 50 127 .+-. 35
118 .+-. 31 polysaccharide with a molecular weight larger than
range of 12,000.about.14,000 The fraction containing 30 125 .+-. 32
97 .+-. 35 polysaccharide with a 100 92 .+-. 25 76 .+-. 30
molecular weight larger than 100,000 The said result derives from
mean .+-. SEM of measurements obtained from the experiments of five
times, five times and six times as for normal group, saline
solution-treated group and the extract-treated group,
respectively.
[0100]
3TABLE 3 The effect of the various stem extract of Acanthopanax
koreanum on the serum level of AST and ALT in liver-injury mice
model induced by D-GalN/LPS. Amount Group (mg/kg) AST ALT Normal
group -- 67 .+-. 11 23 .+-. 11 Physiological saline -- 3456 .+-.
1064 3678 .+-. 1291* solution-treated group(control group) The
water extract-treated 50 598 .+-. 194 634 .+-. 157 group 300 165
.+-. 13 26 .+-. 13 The 70% ethanol extract- 50 2987 .+-. 1859 3125
.+-. 1032 treated group 300 3283 .+-. 1959 3457 .+-. 2373 Treatment
The ethanol 50 446 .+-. 115 464 .+-. 167 of the insoluble part- 300
131 .+-. 11 19 .+-. 11 water treated group extract The ethanol 50
550 .+-. 104 567 .+-. 134 with soluble part- 300 523 .+-. 130 543
.+-. 145 ethanol treated group The fraction containing 50 121 .+-.
14 26 .+-. 11 polysaccharide with a molecular weight larger than
range of 12,000.about.14,000 The fraction containing 30 112 .+-. 15
32 .+-. 12 polysaccharide with a 100 91 .+-. 12 21 .+-. 15
molecular weight larger than 100,000 The said result derives from
mean .+-. SEM of measurements obtained from the experiments of five
times, five times and six times as for normal group, saline
solution-treated group and the extract-treated group,
respectively.
[0101] As shown in the table 2 and 3, the serum level of AST and
ALT in mice model, which administered only saline with D-GalN/LPS
was markedly increased relative to that of the normal group.
[0102] Also, the serum level of AST and ALT in mice model, which
administered the 70% ethanol extract or the supernatant prepared by
treating the water extract with 80% ethanol was similar to that of
the physiological saline solution-treated group. Therefore, it was
shown that the 70% ethanol extract and the supernatant have no
efficiency for the treatment of hepatitis.
[0103] However, the serum level of AST and ALT in mice model, which
administered 50 mg/kg of the water extract was decreased greatly
relative to that of the physiological saline solution-treated
group. The serum level of AST and ALT in mice model, which
administered the 80%-ethanol insoluble part was more decreased.
More preferably, the serum level of AST and ALT in mice model,
which administered 50 mg/kg of the fraction containing
polysaccharide with a molecular weight larger than range of
12,000.about.14,000, among the ethanol insoluble part was the
lowest one of all, and was similar to the that of normal group. The
said fraction has excellent efficacy for treatment of
hepatitis.
EXPERIMENTAL EXAMPLE 3
[0104] The Serum Level of TNF-.alpha. of the Extract on the
Liver-Injury Mice Model Induced by D-GalN/LPS
[0105] The experiment measuring the serum level of TNF-.alpha.,
direct mediator of acute hepatitis in liver-injury mice model
induced by D-GalN/LPS was accomplished as shown in the below.
[0106] After mice (B57BL/6) having body weights of 20 g were
adjusted to a new environment for 1 week, they were used in
experiment. Mice were fully fed before the experiment. The
experiment was accomplished with mice divided to three groups such
as normal group, group to which physiological saline solution was
administered (physiological saline solution-treated group), group
to which the extract was administered (the extract-treated group).
The extract was administered to the mice in the same method as the
experimental example 2.
[0107] The said extract dissolved in physiological saline solution
was intraperitoneally administered to the mice in an amount of 50
mg/kg or 300 mg/kg for two times, respectively. Thereafter D-Gal
and LPS were subsequently administered to the mice in an amount of
700 mg/kg and 10 mg/kg.
[0108] Physiological saline solution was administered to the mice
in the same amount. Blood was collected from mice at 1 hour after
administration. The collected blood was kept at the room
temperature for 1 hour and centrifuged, the serum was isolated. The
prepared serum and liver were stored at -20.degree. C.
[0109] The serum level of TNF-.alpha. was measured by using
enzyme-linked immunosorbent assay (ELISA) kit. The result was shown
in table 4 and 5.
4TABLE 4 The effect of various extracts of Acanthopanax koreanum
root on the serum level of TNF-.alpha. in liver-injury mice model
induced by D-GalN/LPS. Group Amount(mg/kg) TNF-.alpha. (mg/kg)
Normal group -- 26 .+-. 13 Physiological saline solution- -- 678
.+-. 29 treated group(control group) The water extract-treated
group 50 124 .+-. 36 300 74 .+-. 26 The 70% ethanol extract-treated
50 648 .+-. 104 group 300 587 .+-. 87 Treating the The ethanol 50
102 .+-. 26 water extract insoluble part- 300 59 .+-. 15 with
ethanol treated group The ethanol 50 605 .+-. 92 soluble part- 300
260 .+-. 45 treated group The fraction containing 50 32 .+-. 12
polysaccharide with a molecular weight larger than range of
12,000.about.14,000 The fraction containing 30 45 .+-. 21
polysaccharide with a molecular 100 29 .+-. 12 weight larger than
100,000 The said result derives from mean .+-. SEM of measurements
obtained from the experiments of five times, five times and six
times as for normal group, physiological saline solution-treated
group and the extract-treated group, respectively.
[0110]
5TABLE 5 The effect of various extracts of Acanthopanax koreanum
stem on the serum level of TNF-.alpha. in liver-injury mice model
induced by D-GalN/LPS. Group Amount(mg/kg) TNF-.alpha. (mg/kg)
Normal group -- 26 .+-. 11 Physiological saline solution- -- 785
.+-. 17 treated group(control group) The water extract-treated
group 50 132 .+-. 28 300 67 .+-. 16 The 70% ethanol extract-treated
50 690 .+-. 110 group 300 678 .+-. 54 Treating the The ethanol 50
62 .+-. 16 water extract insoluble part- 300 566 .+-. 55 with
ethanol treated group The ethanol 50 233 .+-. 43 soluble part- 300
28 .+-. 11 treated group The fraction containing 50 32 .+-. 12
polysaccharide with a molecular weight larger than range of
12,000.about.14,000 The fraction containing 30 32 .+-. 12
polysaccharide with a molecular 100 25 .+-. 11 weight larger than
100,000 The said result derives from mean .+-. SEM of measurements
obtained from the experiments of five times, five times and six
times as for normal group, saline solution-treated group and the
extract-treated group, respectively.
[0111] As shown in the table 4 and 5, the serum level of
TNF-.alpha. in mice to which D-GalN/LPS was only administered
except the extract was increased thirty times as much as that of
the normal group.
[0112] The serum level of TNF-.alpha. in mice to which 300 mg/kg of
the water extract was administered was decreased.
[0113] More preferably, the serum level of TNF-.alpha. in mice to
which the 80%-ethanol insoluble part of the water extract was
administered was the most similar to that of normal group. The said
80%-ethanol insoluble part has an excellent efficacy for treatment
of hepatitis.
EXPERIMENTAL EXAMPLE 4
[0114] Effect of the Extract on the Survival of the Liver-Injury
Mice Model Induced by D-GalN/LPS
[0115] The experiment measuring survival of liver-injury mice model
induced by D-GalN/LPS was accomplished for 24 hours as shown in the
below.
[0116] After mice (B57BL/6) having body weights of 20 g were
adjusted to a new environment for 1 week with normal diet, they
were used in experiment. Mice were starved for twenty hours before
the experiment. Thereafter, 700 mg/kg of D-Gal and 10 mg/kg of LPS
were administered to the mice. Liver-injury mice model induced by
D-GalN/LPS was prepared for the experiment. The experiment was
accomplished with mice divided to three groups such as normal
group, group to which saline solution was administered, group to
which the extract was administered. The said extract is the water
extract obtained by treating the root or stem of Acanthopanax
Koreanum with water, the 80%-ethanol insoluble part among the said
water extract, or the fraction containing polysaccharide with a
molecular weight larger than 100,000 among the said 80%-ethanol
insoluble part.
[0117] Physiological saline solution was administered to the mice
in the same amount.
[0118] 1. The Effect of the said Water Extract or the said
80%-Ethanol Insoluble Part on the Lethality Rate of the Mice.
[0119] The said water extract or the said 80%-ethanol insoluble
part dissolved in physiological saline solution was administered to
the mice in an amount of 50 mg/kg or 300 mg/kg, respectively.
Thereafter, lethality rate was measured for 24 hours after
administration.
[0120] FIG. 3 shows that the water extract obtained from the root
of Acanthopanax koreanum decreased the lethality of the mice
induced by D-GalN/LPS. Group to which physiological saline solution
was administered was diseased at 6 hours after D-GalN/LPS. All of
the group (eight mice) was diseased within 24 hours.
[0121] However, seven of ten mice to which 300 mg/kg of the water
extract was administered survived within 24 hours, then the
survival rate resulted in 80%. Nine of ten mice to which 300 mg/kg
of the said 80%-ethanol insoluble part was administered survived
within 24 hours, then the survival rate resulted in 90%.
[0122] FIG. 4 shows that the water extract obtained from the stem
of Acanthopanax koreanum decreased the lethality of the mice
induced by D-GalN/LPS. Group to which physiological saline solution
was administered was diseased at 6 hours after D-GalN/LPS. Nine
mice of the group (ten mice) were diseased within 24 hours.
[0123] However, seven of ten mice to which 300 mg/kg of the water
extract was administered survived within 24 hours, then the
survival rate resulted in 70%. Nine of ten mice to which 300 mg/kg
of the said 80%-ethanol insoluble part was administered survived
within 24 hours, then the survival rate resulted in 90%.
[0124] The said result shows that the said water extract or the
said ethanol insoluble part obtained from the root or stem of
Acanthopanax koreanum made the survival rate maintained in the
range of 70%.about.90%. Therefore, the water extract or the
80%-ethanol insoluble part has an efficacy for a liver
protection.
[0125] 2. The Effect of the Polysacccharide Fraction with Molecular
Weight Larger than 100,000 on the Lethality Rate of the Mice.
[0126] The water extract dissolved in physiological saline solution
was intraperitoneally administered to the mice in an amount of 30
mg/kg or 100 mg/kg for two times, respectively. Thereafter,
lethality rate was measured after administration.
[0127] 700 mg/kg of D-GalN and 10 mg/kg of LPS was administered to
the mice, thereafter the fraction containing polysaccharide with a
molecular weight larger than 100,000, among the ethanol insoluble
part was administered to the mice in an amount of 30 mg/kg or 100
mg/kg, respectively. Thereafter, lethality rate was measured for 24
hours after administration.
[0128] FIG. 5 shows that the water extract obtained from the stem
of Acanthopanax koreanum decreased the lethality rate of the mice
induced by D-GalN/LPS. Group to which physiological saline solution
was administered was diseased at 5 hours after administration of
D-GalN/LPS. Also, lethality rate reached 84% (ten of twelve mice
were diseased.) at 6 hours after administration. Lethality rate
reached 92% (eleven of twelve mice were diseased.) at 15 hours
after administration.
[0129] However, lethality rate of mice to which 30 mg/kg or 100
mg/kg of the fraction containing polysaccharide with a molecular
weight larger than 100,000, obtained from the stem of Acanthopanax
koreanum was administered reached 16% (two of twelve mice were
diseased.) or 8% (one of twelve mice were diseased.) at 24 hours
after administration, respectively. Therefore, the fraction has an
efficacy for a liver protection.
[0130] As shown in FIG. 6, mice to which the said fraction was not
administered was diseased at 5 hours after administration of
D-GalN/LPS. Also, lethality rate reached 86% (six of seven mice
were diseased.) at 6 hours after administration. Lethality rate
reached 100% (all of twelve mice were diseased.) at 7 hours after
administration.
[0131] However, lethality rate of mice to which 30 mg/kg or 100
mg/kg of the fraction containing polysaccharide with a molecular
weight larger than 100,000, obtained from the stem of Acanthopanax
koreanum was administered reached 0% (all of twelve mice were
diseased.).
EXPERIMENTAL EXAMPLE 5
[0132] Effect of the Extract on the Survival of the Liver-Injury
Mice Model Induced by D-GalN/TNF-.alpha.
[0133] To confirm whether liver protection of the Acanthopanax
koreanum is directly connected to the inhibition of LPS-induced
production of TNF-.alpha. or inhibition of signaling pathway
through TNF-.alpha. receptor or not, the experiment was
accomplished at the same procedure of the said experiment with
intravenous administration of TNF-.alpha. in an amount of 15
.mu.g/kg instead of LPS. Therefore, acute liver-injury mice model
induced by D-GalN/TNF-.alpha. were prepared for the experiment.
[0134] For the experiment, the fraction containing polysaccharide
with a molecular weight larger than 100,000, displaying the most
excellent survival in the said experiment 5, was administered to
the said mice. In case of control group, physiological saline
solution was only administered to the mice. Six mice per group were
used for the experiment. TNF-.alpha. was intravenously administered
to the mice and then immediately D-GalN was intraperitoneally
administered.
[0135] The fraction was administered to the mice at 12 hours and 1
hour before administration of D-GalN/TNF-.alpha.. The result was
shown in FIG. 7. Five of six mice of the control group were
diseased at 6 hours after administration of D-GalN/TNF-.alpha.,
then lethality rate reached 83%. However, one of six mice of the
administrative group were diseased to 8 hours after administration
of D-GalN/TNF-.alpha., then lethality rate reach 17%. Therefore,
the fraction containing polysaccharide show the high survival in
mice to which TNF-.alpha. was directly administered.
EXPERIMENTAL EXAMPLE 6
[0136] Measurement of DNA Fragmentation in Liver Cell
[0137] The experiment confirming whether the extract inhibits
apoptosis of liver cell in the liver-injury mice model induced by
D-GalN/LPS was accomplished as shown in the below.
[0138] DNA was isolated from the liver tissue of the mice.
Electrophoresis of the isolated DNA was accomplished, thereafter
electro gel was dyed with ethidium bromide to confirm whether DNA
was fragmented or not.
[0139] FIG. 8 show that electrophoresis of DNA (3) and (4) isolated
from the mice to which 300 mg/kg of the water extract obtained from
the root or stem of Acanthopanax koreanum was administered was
similar to one of DNA (1) isolated from the mice to which
physiological saline solution was administered. Preferably,
electrophoresis of DNA (5) isolated from the mice to which 300
mg/kg of the ethanol insoluble part obtained from the root or stem
of Acanthopanax koreanum was administered shows that the said part
has an efficacy to inhibit DNA fragmentation of liver cell in the
liver-injury mice model induced by D-GalN/LPS. Furthermore, the
fraction containing polysaccharide with a molecular weight larger
than range of 12,000.about.14,000 inhibits the fragmentation of DNA
induced by D-GalN/LPS. Therefore, the extract of Acanthopanax
koreanum of the present invention inhibits the fragmentation of DNA
induced by D-GalN/LPS, thus has an efficacy to inhibit apoptosis of
liver cell.
EXPERIMENTAL EXAMPLE 7
[0140] The Effect of the Fraction Containing Polysaccharide with a
Molecular Weight Larger than 100,000 on the Expression of
Pro-Apoptotic Protein.
[0141] The experiment confirming whether the fraction containing
polysaccharide with a molecular weight larger than 100,000 inhibits
expression of pro-apoptotic proteins such as Bid or Bax, which play
an important role in apoptosis of liver cell by D-GalN/LPS, was
accomplished as shown in the below.
[0142] 700 mg/kg of D-GalN and 10 mg/kg of LPS were
intraperitoneally administered to the mice. The fraction or
physiological saline solution was intraperitoneally administered to
the mice at 12 hours and 1 hour before administration of
D-GalN/LPS. The said fraction was administered to the mice in
amount of 10 mg/kg, 30 mg/kg or 100 mg/kg. Liver cell isolated from
the mice to which the said fraction or physiological saline
solution was administered was added to lysis buffer solution
(prepared by mixing 50 mM Tris-HCl, 1% Nonidet P-40, 1 mM EDTA, 1
mM phenylmethyl sulfonyl fluroride, 1 g/ml leupeptin with 150 mM
NaCl, pH 7.5), then homogenized and centrifuged at 15,000.times.g
for 10 min to prepare crude protein. Total concentration of protein
was measured by using Bradford method. 50 .mu.g of protein was
loaded at 12%.about.15% sodium dodecyl sulfate polyacrylamide gel.
After electrophoration the said gel was transferred to PVDF
membrane (Millipore, Bedford, Mass. USA). The membrane was kept at
the solution prepared by mixing Tris-buffered saline solution and
0.1% Tween 20 (Sigma corp.), then 5% skim milk was added to the
said solution. Primary antibody such as rabbit polyclonal anti-Bax
antibody (Santa Cruz Biochemicals, Santa Cruz, Calif. USA) or
anti-Bid antibody (Santa Cruz Biochemicals, Santa Cruz, Calif. USA)
was incubated in the said membrane. Thereafter, the membrane was
washed with buffered saline for 15 min and three times. Thereafter,
secondary antibody was incubated in the membrane for 1 hour. The
membrane was washed. Expression of protein was measured with
Amersham ECL system (Amersham Pharmacia Biotec, Buckinghamshire,
UK).
[0143] FIG. 9 shows that the fraction containing polysaccharide
with a molecular weight larger than 100,000 inhibited expression of
Bax, pro-apoptotic protein concentration dependently. Also, the
expression of Bid, pro-apoptotic protein in the fraction-treated
group was similar to that of Bid in normal group concentration
dependently. However, in case of the mice to which physiological
saline solution was administered, expression of Bid was decreased
relative to the result of normal group. Therefore, the fraction
containing polysaccharide with a molecular weight larger than
100,000 inhibits activation or expression of pro-apoptotic protein
induced by liver-injury in liver cell. Consequently, the fraction
has an efficacy on the liver protection.
EXPERIMENTAL EXAMPLE 8
[0144] Liver Protection in Oral Administration of the Extract of
Acanthopanax koreanum to the Mice
[0145] The experiment confirming whether the 80%-ethanol insoluble
part among the water extract has an efficiency for liver protection
for oral administration was accomplished as shown in the below.
[0146] The said 80%-ethanol insoluble part was dissolved in water.
The solution was orally fed the mice in an amount of 100 mg/kg or
300 mg/kg per day. D-GalN/LPS was administered to the mice at one
week after administration of the said part. The lethality rate was
measured for 24 hours. As for control group to which the said part
was not administered, the lethality rate was measured for 24
hours.
[0147] As shown in FIG. 10 and 11, the control group was diseased
at 6 hours after administration of D-GalN/LPS. All the mice of the
control group were diseased at 12 hours after administration of
D-GalN/LPS. However, three of six mice to which 300 mg/kg of the
said ethanol insoluble part was administered survived at 24 hours
after administration of D-GalN/LPS.
[0148] The said 80%-ethanol insoluble part inhibits the formation
of TNF-.alpha. and expression of various pro-apoptotic proteins.
Therefore, the said ethanol insoluble part has an efficacy for the
liver protection.
EXPERIMENTAL EXAMPLE 9
[0149] Acute Toxicity in Oral Administration of the Extract of
Acanthopanax koreanum to the Mice
[0150] The experiment confirming whether the extract of the present
invention has acute toxicity or not was accomplished as shown in
the below.
[0151] As for the experimental group comprising of five or six mice
(B57BL/6) having body weights of 20 g, the experiment confirming
whether the extract of the present invention has acute toxicity or
not was accomplished. The 80%-ethanol insoluble part among the
water extract of Acanthopanax koreanum prepared in example 2 was
orally administered to the experimental group in an amount of 2000
mg/kg, thereafter toxification of the experimental group was
observed for 7 days. Particularly, death, clinic symptoms, change
of body weight of the mice were observed after administration of
the said part. Also, hematological test and biochemical test of
blood were accomplished. The mice were autopsied to test
abnormality of abdominal cavity or pleural cavity. In the group to
which the said part was administered, abnormality of change of body
weight, clinic symptoms, result of hematological test, biochemical
test of blood, and autopsy was not observed. Also, the mice were
not diseased.
[0152] The said ethanol insoluble part causes no toxic effect in
all of the mice. LD50 for oral administration is 2000 mg/kg.
Therefore, the said ethanol insoluble part has high stability in
the mice body. Also, the said ethanol insoluble part was safely
administered to the body to protect liver.
[0153] Industrial Applicability
[0154] The said extract of the present invention has efficacy
illustrated in the below.
[0155] First, the present invention provides 1) the water extract
of Acanthopanax koreanum obtained by treating the root or stem of
Acanthopanax koreanum with water, 2) the ethanol insoluble part
among the said water extract, obtained by treating the said water
extract with ethanol, 3) the fraction containing polysaccharide
with a molecular weight larger than range of 12,000.about.14,000,
among the said ethanol insoluble part, or 4) the fraction
containing polysaccharide with a molecular weight larger than
100,000, among the said ethanol insoluble part.
[0156] Second, the extract of the present invention reduces the
serum level of AST, ALT and TNF-.alpha. in liver-injury mice model
induced by D-GalN/LPS to be similar to ones of normal group also,
the extract of the present invention inhibits expression of
pro-apoptotic protein of liver cell activated by TNF-.alpha. Third,
the extract of the present invention inhibits that DNA in liver
cell is cleaved to the small fragments.
[0157] Forth, the extract of the present invention made high
survival rate more than 90%, maintained in the experiment for
measurement of lethality rate progressed for 24 hours. Also, the
extract of the present invention has no toxic effects in
histological test. Therefore, the extract of the present invention
can be used as therapeutic agent or preventor of the hepatitis, or
a liver protective drug.
* * * * *